This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The forces involved in achieving the metaphase arrangement of chromosomes are only beginning to be understood. Cohesin holds the sisters together and pulls inward (Tanaka et al., 2000;Vagnarello et al., 2004). Forces generated by microtubules pull the sister kinetochores apart. In higher eukaryotic cells, there is poleward flux of tubulin, but in yeast, no poleward flux of tubulin has been detected (Maddox et al., 2000;Tanaka et al., 2005). The minus microtubule ends are capped at the spindle pole body, and no dynamics have been detected there (Byers et al., 1978;Maddox et al., 2000). In contrast, the plus ends of the kinetochore microtubules are dynamic. In addition to the dynamics of the microtubules themselves, spindle motors exert forces on the chromosomes and the spindle. How the spindle proteins work together to establish metaphase is not known. Recent modeling has shown that dynamic instability of the microtubule plus ends cannot alone produce the kinetochore arrangement achieved by yeast cells in metaphase (Sprague et al., 2003). Our research has provided insight into a molecular basis for the regulation of spindle dynamics. DAM1, a member of the outer kinetochore complex, forms rings that bind microtubules and stabilizes them in vitro (Miranda et al., 2005;Westermann et al., 2005). Vik1 is an accessory protein for the kinesin Kar3 (Manning et al., 1999). We are studying the microtubules at metaphase and anaphase in a DAM1-765 mutant and in the Vik1 mutants, which were isolated in our screen for mutations that affect spindle dynamics. We have already characterized the microtubules in the DAM1-765 mutant by fluorescence microscopy and by fluorescence recovery after photo bleaching. The distribution of tubulin fluorescence is altered in the mutant. We infer from this result that kinetochore microtubules are unusually long and extend across the spindle in this mutant, but only tomographic reconstruction of the spindles can detect the presence of long microtubules and test this prediction. We are now using 3-D EM to examine the arrangement of microtubules in these mutant strains to compare them quantitatively with microtubules in wild type spindles. Preliminary data shows that the microtubules are indeed longer in the mutants, as predict. In future work we will quantify the number of microtubules and map the positions of their ends. Localization of the capped minus ends and the flared plus ends should demonstrate whether the majority of microtubules in the mutant do in fact extend across the length of the spindle.